Original Article Protective effects of traditional Chinese herbal decoction Baihe Dihuang Tang on serum-deprived PC12 cells
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Int J Clin Exp Med 2019;12(8):9899-9907
www.ijcem.com /ISSN:1940-5901/IJCEM0094801
Original Article
Protective effects of traditional Chinese herbal
decoction Baihe Dihuang Tang on
serum-deprived PC12 cells
Min Xing1*, Gang Chen1*, Wenlie Chen1,2,3, Jingjie Mao1,2, Ruhui Lin1,2, Zuanfang Li1,2,3, Jianwei Zeng1,2,3,
Yunmei Huang1,2,3, Liangpu Zheng1,2, Wei Lin1,2, Chunjiang Tan1,2
1
Academy of Integrated Chinese and Western Medicine, 2Fujian Key Laboratory of Integrative Medicine on
Geriatrics, 3National Laboratory of Traditional Chinese Medicine Pharmacology (Cell Structure and Function),
Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, China. *Equal contributors.
Received April 2, 2019; Accepted July 9, 2019; Epub August 15, 2019; Published August 30, 2019
Abstract: Objective: This study aimed to explore the neuroprotective effect of Baihe Dihuang Tang (BDT), a tradition-
al Chinese herbal decoction used for nervous-mental system diseases, on serum-deprived PC12 cell. Methods: BDT
treatment time and concentration were determined through 4,5-dimethylthiazol-2-yl,2,5-diphenyl tetrazolium (MTT)
assay. PC12 cells were randomly divided into three groups: control (cells cultured in serum-containing medium),
model (cells cultured in serum-deprived medium), and BDT (cells cultured in serum-deprived medium and treated
with 3 mg/mL BDT for 24 h) groups. Cell morphology was observed under an inverted phase contrast microscope.
The ultrastructure of cells was studied under a transmission electron microscopy. Membrane potential was deter-
mined using a laser scanning confocal microscope. Total protein and ATP levels were analyzed by the bicinchoninic
acid method and firefly luciferase method, respectively. Results: Compared with the model group, PC12 cells in the
BDT group exhibited extensive morphology and adhered to the culture plate. The ultrastructure was modified and
possessed a smooth membrane and nuclear surface, increased microvilli-like membrane protrusions, homoge-
neously distributed organelles, rare pinocytosis and phagocytosis, abundant ribosomes, rare vacuoles, and normal-
shaped nucleus. BDT also enhanced the total cell protein content, ATP level, and membrane hyperpolarization.
Conclusion: BDT could reduce cell death and neural excitability and regulate energy metabolism caused by serum
deprivation. We suggest that BDT could modify the hypometabolic state and neural overactivity in chronic fatigue
syndrome (CFS). This work may provide new insights into the possibility of using BDT as a therapeutic agent for CFS.
Keywords: Baihe Dihuang Tang, PC12 cell, ultrastructure, cellular membrane potential, chronic fatigue syndrome
Introduction comes red [1]”. BDT consists of Lilii Bulbus
(Baihe) and Rehmanniae Radix (Dihuang). In-
Baihe Dihuang Tang (BDT) was first recorded tegrative pharmacology studies predicted that
in “Synopsis of Prescriptions of the Golden the action of BDT may be related to regulating
Chamber” (Jinkui Yaolue) by Zhongjing Zhang, a neurotrophic factors, disorder of amino acids,
prestigious specialist in traditional Chinese and purine metabolism and catabolism [2]. At
medicine (TCM) during 210 A.D. [1]. BDT was present, BDT is a widely used recipe in TCM to
initially used to cure Baihe Bing, also called Lily cure nervous-mental illnesses [3].
disease. According to Zhang, “Baihe Bing is
characterized by general malaise and inability Chronic fatigue syndrome (CFS)/Myalgic Ence-
to eat, talk, lie down, or walk. A patient with this phalomyelitis (ME) is a generalized disease th-
disease often appears quiescent and may so- at persists for more than 6 months. It has
metimes have an appetite, while other times unknown etiology, and manifests as a hetero-
not. The patient feels cold but has no chills or geneous multisystem disorder, including neu-
feels hot but has no fever. A bitter taste dis- rological problems, immune dysfunction, hor-
turbs his/her mouth, and his/her urine be- monal imbalance, gastrointestinal symptoms,Neuroprotective effects of BDT
Figure 1. High‑performance liquid chromatography chromatography. A. Catalpol standard solution. B. Rehmanniae
Radix. C. Baihe Dihuang Tang. mAU: milli-absorbance units.
and joint and muscle pain [4, 5]. CFS occurs in neural activity was determined by measuring
approximately 0.1% to 5% of the world popula- membrane potential. The present results may
tion [6, 7]. Despite the high social burden and possibly reinforce the application of BDT for
heavy economic burden caused by CFS, no spe- CFS.
cific therapy is available. Western medicine of-
fers potent cures for diseases with a single Materials and methods
cause and definite pathophysiology. However,
Preparation of BDT
the etiology of CFS remains unidentified [8].
Thus, only graded exercise therapy and cogni-
BDT used in this study is composed of Lilii
tive behavioral therapy have been shown to be Bulbus (Baihe, the dried bulbs of Lilium lancifo-
effective for CFS [9]. TCM herbal formulas have lium Thunb., 80 g) and Rehmanniae Radix (Di-
multiple targets on a disease with network con- huang, the dried root of Rehmannia glutinosa
nection [10, 11]. A new TCM recipe for CFS Libosch., 60 g) at a ratio of 4:3 [18]. The crude
treatment must be developed. CFS has several drugs were bought from Guo Yi Tang Chinese
similarities to Baihe Bing in terms of symptoms, Herbal Medicine Store (Fuzhou, Fujian, China).
etiology, pathology, and treatment [12, 13]. In The herbs were boiled in 1200 mL of water for
this regard, we speculated that BDT could be a 30 min, and the decoction was filtered. The
potential therapy for CFS. residues were added with 900 mL of water,
boiled, and filtered again. The extracts were
BDT can regulate neurological dysfunction, su-
combined, dried under vacuum, and stored at
ch as depression and psychological suboptimal
-20°C.
health state [14, 15]; however, few works have
examined its effects on CFS. Rat pheochromo- The catalpol in Rehmanniae Radix was proved
cytoma PC12 cells are similar to sympathetic to be neuroprotective [19]. Thus, the catalpol
neurons in terms of morphology and function was used for quantitative analysis of BDT. It
and are widely used for neuronal disease re- was detected by Agilent 1200 high‑perfor-
search, such as in in vitro cell models [16]. mance liquid chromatography system (Agilent,
Patients with CFS are usually under a hypomet- santa Clara, CA, USA) with a C-18 column
abolic survival state [17], so we used serum (4.6×250 mm, 5 µm). The mobile phase con-
deprivation to simulate a similar living condi- sisted of acetonitrile-0.1% phosphoric aq. (1:
tion. The effect of BDT on modifying neuronal 99) at a flow rate of 1 mL/min. The column tem-
metabolism was evaluated by the analysis of perature was 30°C and the detection wave-
cell morphology, ultrastructure, total protein length was 210 nm. The content of catalpol in
content, and ATP level. The effect of BDT on BDT was found to be 0.22% (Figure 1).
9900 Int J Clin Exp Med 2019;12(8):9899-9907Neuroprotective effects of BDT
Cell culture phase contrast microscope at a magnification
of 200×.
Rat PC12 cells were purchased from the Am-
erican Type Culture Collection (Rockville, MD, Cell ultrastructure observation
USA). The cells were incubated at 37°C in an
incubator with 90% humidified atmosphere Cells were collected and fixed by glutaralde-
(Thermo Fisher Scientific, Waltham, MA, USA) hyde and osmium tetroxide, dehydrated with
containing 5% CO2. The cells were then cultured alcohol and acetone, and embedded in epoxy
in RPMI 1640 medium supplemented with 10% resin. The samples were ultrathin sectioned
fetal bovine serum (FBS), 100 U/mL penicil- and stained with uranylacetate and lead citra-
lin, and 100 μg/mL streptomycin. RPMI 1640 te. The cell ultrastructure was observed and
medium, FBS, penicillin, and streptomycin were photographed using a transmission electron
acquired from Hyclone (Logan, UT, USA). microscope (H-7650, Hitachi, Japan).
Cell treatment condition selection Cellular total protein content and ATP level de-
termination
BDT treatment condition was selected with
MTT assay. For selection of BDT treatment Cellular total protein and ATP level analyses
time, PC12 cells were incubated in serum- were conducted using our previously reported
deprived medium for 0, 12, 24, and 48 h. Cell method [20]. Protein content was determined
viability was observed under an inverted phase using a bicinchoninic acid (BCA) protein assay
contrast microscope (IX70, Olympus, Tokyo, kit (Beyotime Institute of Biotechnology, Sh-
Japan). anghai, China). ATP level was measured using a
firefly luciferase-based ATP assay kit (Beyotime
For selection of BDT treatment concentration, Institute of Biotechnology). PC12 cells were
PC12 cells were seeded in 96-well plates at a plated in a 24-well plate. After intervention, the
concentration of 2×105 cells/mL, with 100 μL cells were rinsed with phosphate-buffered sa-
of the cells per well. The cells were then pre- line (pH 7.4, Gibco Life Technologies), disrupted
treated with BDT at various concentrations (0, in lysis buffer (Sigma Aldrich), and centrifuged.
1.5, 3, 6, 12, 24, and 48 mg/mL) for 4 h. Each The supernatant was collected and mixed with
well was added with 4,5-dimethylthiazol-2-yl, BCA protein/ATP detection working solution.
2,5-diphenyl tetrazolium (MTT, Sigma Aldrich, Absorbance was recorded at 562 nm. Standard
St Louis, MO, USA) at 37°C for 4 h. The purple- curve of protein or ATP was generated using
blue MTT formazan precipitate was dissolved the standard. ATP level was expressed as nor-
using dimethylsulfoxide (Sigma Aldrich). Absor- malized luminance (NRLU, in nmol/mg protein)
bance was determined using a microplate re- [21].
ader (Bio-Rad Laboratories, Inc., Hercules, CA,
USA) at 570 nm. Cell membrane potential assay
Cell grouping and treatment Cells were seeded into a six-well plate, added
with bis-(1,3-dibarbituric acid)-trimethine oxa-
After the proper BDT treatment condition was nol [diBAC4(3), Sigma Aldrich], and incubated
selected, PC12 cells were randomly divided at 37°C for 30 min. Cell membrane potential
into three groups. Cells in the control group we- was examined under a laser confocal scanning
re cultured in serum-containing medium. Cells microscope (LSM710, Carl Zeiss Meditec, Jena,
in the model group were cultured in serum- Germany) with laser light at 488 nm.
deprived medium. Cells in the BDT group were
cultured in serum-deprived medium and treat- Statistical analysis
ed with BDT.
SPSS 23.0 (SPSS, Chicago, Illinois, USA) was
Cell morphology observation used for statistical analyses. Quantitative data
are
_ expressed as mean ± standard deviation
PC12 cells were seeded into six-well plates at ( x ± sd) of at least triplicate experiments.
a density of 2×105 cells/mL. Cell morphology Variance among groups were analyzed using
was observed and photographed by an inverted Student’s t-test and one-way analysis of vari-
9901 Int J Clin Exp Med 2019;12(8):9899-9907Neuroprotective effects of BDT
the serum-deprived medi-
um, cells in the model gr-
oup exhibited a degenerat-
ed, shrunk, and round mor-
phology and were detached
from the culture plate (Fi-
gure 3B). After BDT treat-
ment, the cell adherence
rate to the plate and the
cell volume were increased.
This finding indicated the
improved cellular morphol-
ogy of cells in the BDT
Figure 2. Changes in the viability of serum-deprived PC12 cells determined group (Figure 3C). Comp-
through MTT assay. PC12 cells were pretreated with BDT (0, 1.5, 3, 6, 12, 24, ared with cells in the mod-
and 48 mg/mL) for 24 h. Cell survival rate was analyzed with MTT assay. Cell el group, the morphology
viability was expressed as the percentage (%) of cells cultured in medium con- of PC12 cells cultured in
taining 10% fetal bovine serum. Data are presented as mean ± SD (n = 3).
*PNeuroprotective effects of BDT Figure 3. Morphological changes in PC12 cells observed under an inverted phase contrast microscope at magnifi- cation of 200×. A. Cells in the control group were cultured in serum-containing medium. The cells exhibited neurite structure and were attached to the plate. B. Cells in the model group were cultured in serum-deprived medium. The cells exhibited degenerated, shrunk, and round morphology and were detached from the culture plate. C. Cells in the BDT group were cultured in serum-deprived medium and treated with 3 mg/mL BDT for 24 h. The cells adhered to the plate, and the cellular volume was increased. D. PC12 cells cultured in medium containing 10% fetal bovine serum and treated with 3 mg/mL BDT for 24 h. The morphology of these cells was similar to that of cells in the control group. Bar = 10 µm. Figure 4. Ultrastructural changes in PC12 cells determined through transmission electron microscopy. Magnifica- tion 15,000× and 30,000×. A, B. Cells in the control group were cultured in serum-containing medium. Abundant 9903 Int J Clin Exp Med 2019;12(8):9899-9907
Neuroprotective effects of BDT microvilli-like membrane protrusions and ribosomes and scattered distribution of heterochromatin were observed. C, D. Cells in the model group were cultured in serum-deprived medium. The number of microvilli-like membrane protrusions was decreased, and the incidence rates of membrane traffic events including pinocytosis and phagocy- tosis were increased. The ribosomes were dissociated, and their numbers decreased. The number of vacuoles was increased. Partly swollen mitochondria and partly dilatant endoplasmic reticulum were observed. Heterochromatin was condensed and clung to the nuclear envelope, and irregular nuclear borders were observed. E, F. Cells in the BDT group were cultured in serum-deprived medium and treated with 3 mg/mL BDT for 24 h. The occurrence of pinocytosis and phagocytosis was decreased. The ribosomes and mitochondria were abundant. Cell nucleus was spherical, and heterochromatin had scattered distribution. White arrows: Pinocytosis vesicles; Black arrows phago- some; V: vacuoles; RRE: ribosomes in the rough endoplasmic reticulum; H: heterochromatin; M: mitochondrion; G: Golgi complex. Bar = 1 µm. Figure 5. A. Total protein changes in PC12 cells determined through BCA method. B. Cellular ATP levels in the three groups determined through firefly luciferase-based ATP assay. ATP level was expressed as the normalized luminance (NRLU) of firefly luciferase (nmol/mg protein). Data are presented as mean ± SD (n = 3). *P
Neuroprotective effects of BDT Figure 6. Membrane potential changes in PC12 cells. A-C. PC12 cells were stained green by diBAC4 (3) under a laser scanning confocal microscope. A. Cells in the control group were cultured in serum-containing medium. B. Cells in the model group were cultured in serum-deprived medium. C. Cells in the BDT group were cultured in serum-deprived medium and treated with 3 mg/mL BDT for 24 h. D. Histograms represent the fluorescence inten- sity comparison among the three groups. Fluorescence intensity values were obtained through a laser scanning confocal microscope. Data are presented as mean ± SD (n = 3). *P
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